Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

Briefly described, embodiments of this disclosure, among others, include
compositions, gels, methods for synthesizing multifunctional silica based
nanoparticle gel, method of treating, preventing, or both treating and
preventing, a disease in a plant species, method for simultaneously
treating citrus plants for citrus canker and preventing the invasion of
an Asian Citrus Psyllid (ACP) vector that carries the pathogen and
spreads the citrus greening disease in citrus plants, and the like.

Claims:

1. A composition, comprising a multifunctional silica based nanoparticle
including a first component and a second component, wherein the first
component functions as an antibacterial, an antifungal, or a combination
thereof, wherein the second component functions as a repellant for
insects.

2. The composition of claim 1, wherein the first component is selected
from a copper component, a zinc component, a titanium component, a cerium
component, a magnesium component, a zirconium component, a
polyethyleneimine (PEI), a fullerene, a carbon nanotube, and a
combination thereof; and the second component is a sulfur compound.

3. The composition of claim 2, wherein the sulfur component is selected
from: an alkyl sulfide, an alkyl disulfide, an alkyl trisulfide, an alkyl
tetrasulfide, and a combination thereof.

10. The composition of claim 9, wherein the DMDS is attached to the
multifunctional silica based nanoparticle via copper ions.

11. A gel comprising: a plurality of multifunctional silica based
nanoparticles of any one of claims 1 to 10 disposed in an amorphous
silica material, wherein the amorphous silica material includes one or
both of the first component and the second component.

12. The gel of claim 11, further comprising a second silane compound.

13. The gel of claim 11, wherein the second silane compound is selected
from the group consisting of: 3-(trihydroxysilyl) propyl
methylphosphonate, alkyl silane, tetraethoxysilane, tetramethoxysilane,
sodium silicate, a silane precursor that can produce silicic acid or
silicic acid like intermediates, and a combination thereof.

14. A method for synthesizing a multifunctional silica based nanoparticle
gel, comprising: adding a portion of a loaded silica nanoparticle to an
aqueous reaction medium to form mixture I; adding a portion of a second
component directly to the aqueous reaction medium containing mixture I to
form mixture II; and mixing mixture II to form a multifunctional silica
nanoparticle gel that includes multifunctional silica nanoparticles of
any one of claims 1 to 10.

15. A method for synthesizing a multifunctional silica based nanoparticle
gel, comprising: adding a portion of a powdered loaded silica
nanoparticle to a reaction vessel; adding a portion of a second component
directly to the powder to form mixture A; and mixing mixture A to form a
multifunctional silica nanoparticle gel that includes multifunctional
silica nanoparticles of any one of claims 1 to 10.

16. A method of treating, preventing, or both treating and preventing, a
disease in a plant species, comprising: administering a composition
including multifunctional silica based nanoparticles of claims 1 to 10,
multifunctional silica based nanoparticle gel of claims 10 to 13, or a
mixture thereof, to a the plant.

17. The method of claim 16, wherein the plant treated is a member of the
citrus species.

18. The method of claim 17, wherein the disease treated is selected from
the group consisting of citrus canker, citrus greening, and a combination
thereof.

19. The method of claim 16, wherein the first component, the second
component, or both the first component and second component are released
in a sustained manner for treatment of the plant for a time period from
the day the composition is administered to about eight months.

20. A method for simultaneously treating citrus plants for citrus canker
and preventing the invasion of an Asian Citrus Psyllid (ACP) vector that
carries the pathogen and spreads the citrus greening disease in citrus
plants, comprising the steps of: administering a composition including
multifunctional silica based nanoparticles of claims 1 to 10,
multifunctional silica based nanoparticle gel of claims 10 to 13, or a
mixture thereof, to a the citrus plant, wherein administering includes
substantially covering the leaves and branches of the citrus plant.

21. The method of claim 20, further comprising; allowing the leaching of
Cu ions and dimethyl disulfide (DMDS) in a slow-release, non-phytotoxic
manner with exposure to atmospheric conditions selected from at least one
of rain, wind, snow, and sunlight, wherein a plurality of copper ions and
dimethyl disulfide diffuse out from a location of application on a plant
tissue surface and cover the exposed tissue surface of rapidly growing
fruit and leaves, thereby minimizing the application frequency per
growing season, increasing plant surface coverage and longevity of
coverage of citrus plants requiring treatment for citrus canker and
citrus greening disease.

22. The method of claim 20, wherein the silica based formulation further
includes a second silane compound to achieve uniform plant surface
coverage to modulate copper ion release rate, improve rain-fastness and
increase longevity of plant surface coverage.

23. The method of claim 22, wherein the silane precursor compound is
selected from the group consisting of: 3-(trihydroxysilyl) propyl
methylphosphonate, alkyl silane, tetraethoxysilane, tetramethoxysilane,
sodium silicate, a silane precursor that can produce silicic acid or
silicic acid like intermediates, and a combination thereof.

[0003] The worldwide citrus industry is currently battling with two
potentially devastating diseases: citrus greening, also known as
Huanglongbing or HLB, and citrus canker.

[0004] Citrus greening is the most destructive, highly infectious disease
of most commercial citrus varieties. This disease is caused by the
Gram-negative HLB bacterium that belongs to the genus Candidatus
Liberibacter as reported by J. M. Bove in "Huanglongbing: A destructive,
newly-emerging, century-old disease of citrus," Journal of Plant
Pathology 2006, 88, (1), 7-37. HLB threatens the citrus industry
worldwide and may cause damage to the citrus industry and economy in the
State of Florida. The problem can be severe and kill citrus trees. The
disease causes fruits to taste bitter and become deformed, small-sized
and poorly-colored, making it unusable and unmarketable. Currently, there
is no cure for the HLB.

[0005] The Asian Citrus Psyllid (ACP) is an invasive phloem-feeding insect
that causes serious damage to citrus plants and citrus plant relatives.
Burned tips and twisted leaves result from an infestation of ACP on new
growth. In addition, ACP is a vector of HLB and carries HLB and can
rapidly spread the disease from one grove to another.

[0007] Unfortunately, the use of foliar insecticides appears to be the
only solution available to growers these days to prevent HLB infection,
even though such integrated practices are expensive and labor extensive.

[0009] While there is no cure for the HLB disease, canker losses have been
controlled by the use of appropriate anti-bacterial agents such as copper
(Cu) based compounds, including, but not limited to, Cu oxychloride, Cu
sulphate, Cu hydroxide, Cu oxide, ammonia-Cu carbonate, antibiotics, such
as, streptomycin, tetracycline, and induced systemic resistance
compounds, including, acibenzolar-S-methyl, harpin protein. To date,
copper (Cu) has been the gold standard for controlling citrus canker
disease worldwide due to its effectiveness in protecting against the
possibility of infection and minimal development of Cu resistance by a
pathogen.

[0010] Due to destructive nature of HLB and citrus canker diseases, there
is a need to find solutions to combat HLB and citrus canker diseases.

SUMMARY

[0011] Briefly described, embodiments of this disclosure, among others,
include compositions, gels, methods for synthesizing multifunctional
silica based nanoparticle gel, method of treating, preventing, or both
treating and preventing, a disease in a plant species, method for
simultaneously treating citrus plants for citrus canker and preventing
the invasion of an Asian Citrus Psyllid (ACP) vector that carries the
pathogen and spreads the citrus greening disease in citrus plants, and
the like.

[0012] An exemplar embodiment of a composition, among others, includes a
multifunctional silica based nanoparticle including a first component and
a second component, wherein the first component functions as an
antibacterial, an antifungal, or a combination thereof, wherein the
second component functions as a repellant for insects.

[0013] An exemplar embodiment of a gel, among others, includes, a
plurality of multifunctional silica based nanoparticles as described
herein are disposed in an amorphous silica material, wherein the
amorphous silica material includes one or both of the first component and
the second component.

[0014] An exemplar embodiment of a method for synthesizing multifunctional
silica based nanoparticle gel, among others, includes, adding a portion
of loaded silica nanoparticle to an aqueous reaction medium to form
mixture I; adding a portion of a second component directly to the aqueous
reaction medium containing mixture Ito form mixture II; and mixing
mixture II to form a multifunctional silica nanoparticle gel that
includes multifunctional silica nanoparticles as described herein.

[0015] An exemplar embodiment of a method for synthesizing multifunctional
silica based nanoparticle gel, among others, includes, adding a portion
of powdered loaded silica nanoparticle to a reaction vessel; adding a
portion of a second component directly to the powder to form mixture A;
and mixing mixture A to form a multifunctional silica nanoparticle gel
that includes multifunctional silica nanoparticles as described herein.

[0016] An exemplar embodiment of a method of treating, preventing, or both
treating and preventing, a disease in a plant species, among others,
includes, administering a composition including multifunctional silica
based nanoparticles as described herein, multifunctional silica based
nanoparticle gel as described herein, or a mixture thereof, to a the
plant.

[0017] An exemplar embodiment of a method for simultaneously treating
citrus plants for citrus canker and preventing the invasion of an Asian
Citrus Psyllid (ACP) vector that carries the pathogen and spreads the
citrus greening disease in citrus plants, among others, includes,
administering a composition including multifunctional silica based
nanoparticles as described herein, multifunctional silica based
nanoparticle gel as described herein, or a mixture thereof, to a the
citrus plant, wherein administering includes covering the leaves and
branches of the citrus plant.

[0018] Other compositions, gels, methods, features, and advantages of this
disclosure will be or become apparent to one with skill in the art upon
examination of the following drawings and detailed description. It is
intended that all such additional apparatuses, systems, methods,
features, and advantages be included within this description, be within
the scope of this disclosure, and be protected by the accompanying
claims.

[0023]FIG. 3B is a plot of gas chromatography-mass spectroscopy (GC-MS)
data of a chloroform extract of DMDS added to dry SiNG as a control
showing no DMDS peaks.

DETAILED DESCRIPTION

[0024] Before the present disclosure is described in greater detail, it is
to be understood that this disclosure is not limited to particular
embodiments described, and as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present disclosure will be limited only
by the appended claims.

[0025] Where a range of values is provided, it is understood that each
intervening value, to the tenth of the unit of the lower limit unless the
context clearly dictates otherwise, between the upper and lower limit of
that range and any other stated or intervening value in that stated
range, is encompassed within the disclosure. The upper and lower limits
of these smaller ranges may independently be included in the smaller
ranges and are also encompassed within the disclosure, subject to any
specifically excluded limit in the stated range. Where the stated range
includes one or both of the limits, ranges excluding either or both of
those included limits are also included in the disclosure.

[0026] Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this disclosure belongs. Although any methods
and materials similar or equivalent to those described herein can also be
used in the practice or testing of the present disclosure, the preferred
methods and materials are now described.

[0027] All publications and patents cited in this specification are herein
incorporated by reference as if each individual publication or patent
were specifically and individually indicated to be incorporated by
reference and are incorporated herein by reference to disclose and
describe the methods and/or materials in connection with which the
publications are cited. The citation of any publication is for its
disclosure prior to the filing date and should not be construed as an
admission that the present disclosure is not entitled to antedate such
publication by virtue of prior disclosure. Further, the dates of
publication provided could be different from the actual publication dates
that may need to be independently confirmed.

[0028] As will be apparent to those of skill in the art upon reading this
disclosure, each of the individual embodiments described and illustrated
herein has discrete components and features which may be readily
separated from or combined with the features of any of the other several
embodiments without departing from the scope or spirit of the present
disclosure. Any recited method can be carried out in the order of events
recited or in any other order that is logically possible.

[0029] Embodiments of the present disclosure will employ, unless otherwise
indicated, techniques of chemistry, botany, biology, and the like, which
are within the skill of the art.

[0030] The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and description of
how to perform the methods and use the probes disclosed and claimed
herein. Efforts have been made to ensure accuracy with respect to numbers
(e.g., amounts, temperature, etc.), but some errors and deviations should
be accounted for. Unless indicated otherwise, parts are parts by weight,
temperature is in ° C., and pressure is at or near atmospheric.
Standard temperature and pressure are defined as 20° C. and 1
atmosphere.

[0031] Before the embodiments of the present disclosure are described in
detail, it is to be understood that, unless otherwise indicated, the
present disclosure is not limited to particular materials, reagents,
reaction materials, manufacturing processes, or the like, as such can
vary. It is also to be understood that the terminology used herein is for
purposes of describing particular embodiments only, and is not intended
to be limiting. It is also possible in the present disclosure that steps
can be executed in different sequence where this is logically possible.

[0032] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a compound" includes a plurality of compounds. In
this specification and in the claims that follow, reference will be made
to a number of terms that shall be defined to have the following meanings
unless a contrary intention is apparent.

DEFINITIONS AND ABBREVIATIONS

[0033] Si is used herein to mean silicon dioxide, which is also commonly
known as "silica."

[0034] NG stands for "Nanogel", which is the gel-like substance formed by
the interconnection of nanoparticles, for example, the interconnection of
multifunctional silica based nanoparticles.

[0035] NP stands for "Nanoparticle", which can have a particle size (e.g.,
diameter for spherical or substantially spherical nanoparticles) of about
10 to 500 nm, about 10 to 250 nm, about 10 to 100, or about 10 nm to 50
nm. The diameter can be varied from a few nanometers to hundreds of
nanometers by appropriately adjusting synthesis parameters, such as
amounts of silane precursor, amounts of hydrolyzing agents, polarity of
reaction medium, and the like.

[0036] CuSiNP stands for copper loaded silica nanoparticle.

[0037] CuSiNG stands for copper loaded silica nanogel.

[0038] HCuSiNG stands for hybrid Cu-loaded silica nanogel, where the SiNG
matrix is loaded with a second silane compound to achieve uniform or
substantially uniform plant surface coverage.

[0040] "Uniform plant surface coverage" refers to a uniform and complete
(e.g., about 100%) wet surface due to spray application of embodiments of
the present disclosure. In other words, spray application causes
embodiments of the present disclosure to spread throughout the plant
surface.

[0041] "Substantial uniform plant surface coverage" refers to about 70%,
about 80%, about 90%, or more uniform plant surface coverage.

[0042] "Substantially covering" refers to covering about 70%, about 80%,
about 90%, or more, of the leaves and branches of a plant.

[0043] "Plant" refers to trees, plants, shrubs, flowers, and the like as
well as portions of the plant such as twigs, leaves, stems, and the like.
In a particular embodiment, the term plant includes a fruit tree such as
a citrus tree (e.g., orange tree, lemon tree, lime tree, and the like).

[0045] As used herein, "treat", "treatment", "treating", and the like
refer to acting upon a disease or condition with a multifunctional silica
based nanoparticle or gel of the present disclosure to affect the disease
or condition by improving or altering it. In addition, "treatment"
includes completely or partially preventing (e.g., about 70% or more,
about 80% or more, about 90% or more, about 95% or more, or about 99% or
more) a plant form acquiring a disease or condition. The phrase "prevent"
can be used instead of treatment for this meaning. "Treatment," as used
herein, covers one or more treatments of a disease in a plant, and
includes: (a) reducing the risk of occurrence of the disease in a plant
predisposed to the disease but not yet diagnosed as infected with the
disease (b) impeding the development of the disease, and/or (c) relieving
the disease, e.g., causing regression of the disease and/or relieving one
or more disease symptoms.

[0046] The term "antibacterial" refers to a compound or composition that
destroys bacteria, suppresses or prevents bacteria growth, and/or
suppresses, prevents or eliminates the ability of bacteria to reproduce.

[0047] The term "antifungal" refers to a compound or composition that
destroys fungus, suppresses or prevents fungus growth, and/or suppresses,
prevents or eliminates the ability of the fungus to reproduce.

Discussion:

[0048] Embodiments of the present disclosure include multifunctional
silica based nanoparticles, methods of making multifunctional silica
based nanoparticles, multifunctional silica based nanoparticle gels,
methods of making multifunctional silica based nanoparticle gels, methods
of using methods of making multifunctional silica based nanoparticle and
methods of making multifunctional silica based nanoparticle gels, methods
of treating plants, and the like. Embodiments of the present disclosure
provide for a composition that can be used for multiple purposes.
Embodiments of the present disclosure are advantageous in that they can
slowly release one or more agents that can be used to prevent or
substantially prevent and/or treat or substantially treat a disease or
condition in a plant, act as an antibacterial and/or antifungal, and/or
act as a repellent to certain types of insects. Another advantage of an
embodiment of the present disclosure is that the agent(s) can be
controllably released over a long period of time (e.g., from the day of
application until a few weeks or months (e.g., about 6 or 8 months)).
Embodiments of the present disclosure and features and advantages of
these embodiments will be discussed in further detail herein.

[0049] Embodiments of the multifunctional silica based nanoparticle can
include a first component and a second component. In addition,
embodiments of the present disclosure can include a multifunctional
silica based nanoparticle gel that includes multifunctional silica based
nanoparticles. The multifunctional silica based nanoparticle and/or
multifunctional silica based nanoparticle gel can be included in a
composition to be administered (e.g., spraying) to a plant. The
multifunctional silica based nanoparticle gel includes multifunctional
silica based nanoparticles inter-connected in an amorphous silica
material. The nanoparticles can be inter-connected covalently (e.g.,
through --Si--O--Si-- bonds), physically associated via Van der Waal
forces, and/or through ionic interactions (e.g., positively charged
copper ions and negatively charged silica nanoparticles). The first and
second components can be within the amorphous material as well as in the
multifunctional silica based nanoparticle.

[0050] The first component can function as an antibacterial and/or
antifungal, specifically, treating, substantially treating, preventing or
substantially preventing, plant diseases such as citrus greening (HLB)
and citrus canker diseases. The first component (e.g., Cu) can be
released from the multifunctional silica based nanoparticle or gel so
that it can act as an antibacterial and/or antifungal for a period of
time (e.g., from application to days to months). The second component
functions as a repellant for insects that can harm plants and/or carry
bacteria, diseases, fungi, and the like, that can harm plants (e.g.,
fruit tree). The second component (e.g., a sulfur compound) of the
multifunctional silica based nanoparticle or gel can act as an Asian
Citrus Psyllid (ACP) repellant for a period of time (e.g., from
application to days to months). Embodiments of the present disclosure
have multifunctional purposes to combat diseases in plants such as trees,
bushes, and the like, for example, simultaneously treating, substantially
treating, preventing and/or substantially preventing citrus greening and
citrus canker diseases.

[0051] In an embodiment, the release rate of the first and/or second
component can be controlled so that characteristics of one or both can be
effective for time frames of days to weeks or to months. In other words,
the first component and/or second component can be released from the
multifunctional silica based nanoparticle or gel starting from the day of
application and continuing release to about a week, about a month, about
two months, about three months, about four months, about five months,
about six months, about seven month, or about eight months.

[0052] The multifunctional silica based nanoparticle includes a core and
shell. The core includes silica loaded with a first type of first
component (e.g., Cu ions). The silica shell that can support a second
type of first component (e.g., copper oxide), while also including the
first component (e.g., Cu ions). Although not intending to be bound by
theory, in an embodiment the second component can interact with the first
component (e.g., Cu ions) via a charge-transfer type of interaction,
where interaction between the first component and the second component
can occur in the nanoparticle and/or in the amorphous silica material.

[0054] In an embodiment, the copper component can include a copper ion,
metallic copper, copper oxide, copper oxychloride, copper sulfate, copper
hydroxide, and a combination thereof. The copper component can include
copper ions that are electrostatically bound to the silica nanoparticle
core or amorphous silica matrix, copper covalently bound to the hydrated
surface of the nanoparticle or amorphous silica matrix, and/or copper
oxides and/or hydroxides bound to the surface of the nanoparticle or
amorphous silica matrix. In an embodiment, the multifunctional silica
based nanoparticle and/or gel includes the copper component in two or in
all three of these states.

[0055] In an embodiment, the copper component can be in a soluble
(amorphous) and an insoluble (crystalline) form. By controlling the
soluble and insoluble ratio, the release rate of the copper component can
be controlled as a function of time. As a result, the release rate of the
copper component can be controlled so that antibacterial and/or
antifungal characteristics can be effective for time frames of days to
weeks or to months. In other words, the copper component can be released
from the multifunctional silica based nanoparticle or gel staring from
the day of application and continuing release to about a week, about a
month, about two months, about three months, about four months, about
five months, about six months, about seven month, or about eight months.
The ratio of the soluble to insoluble copper component can be adjusted to
control the release rate. In an embodiment, the ratio of the soluble
copper to the insoluble copper (e.g., Chelated Cu)X (Crystalline
Cu)1-X) can be out 0:1 to 1:0, and can be modified in increments of
about 0.01 to produce the ratio that releases the Cu for the desired
period of time. Parameters that can be used to adjust the ratio include:
solvent polarity and protic nature (i.e., hydrogen bonding capability),
Cu precursor (e.g., Cu sulfate) concentration, temperature, concentration
of silane precursor (such as tetraethylorthosilicate, TEOS), and the
like.

[0056] In an embodiment, the second component can include a sulfur
compound. The sulfur compound does not react or reacts very little (e.g.,
at such a low percentage or at such a slow rate that the first and second
components can still function in a manner and for a time frame described
herein) with the first component. The sulfur compound can include alkyl
sulfides, alkyl disulfides, alkyl trisulfides, alkyl tetrasulfides,
analogues of each, and a combination thereof, where alkyl can include
alkyl, dialkyl, and trialkyl. In particular, the sulfur compound can
include compounds dimethyl disulfide (DMDS), dimethyl sulfide, diethyl
disulfide, diethyl trisulfide, diethyl tetrasulfide, and a combination
thereof. The release rate of the second component can be controlled to
release starting from the day of application to about a week, about a
month, about two months, about three months, about four months, about
five months, about six months, about seven month, or about eight months.

[0057] In an embodiment, the sulfur compound is DMDS. It should be noted
that the sulfur compound can be used as a ACP repellant and is attractive
strategy to control the HLB. It should be noted that DMDS is toxic to
insects because it disrupts cytochrome oxidase system of the mitochondria
and is considered a strong repellent to ACP. DMDS can interact with the
first component (e.g., Cu ion) in a charge-transfer type of interaction.
Thus, by controlling the amount of first component that the DMDS can
interact with, the amount of the first component can be used to control
the amount of DMDS present in the multifunctional silica based
nanoparticle or gel. Although not intending to be bound by theory, sulfur
(electronegative element, polarizable) in DMDS is weakly bound to copper
ions (type of ion-dipole interaction). Once Cu is released from the
product, DMDS will mostly release as there is no other strong interaction
between DMDS and silica nanoparticle/nanogel matrix other than Van der
Waals force.

[0058] In an embodiment, the amorphous silica gel has no ordered (e.g.,
defined) structure (opposite to crystalline structure) so an "amorphous
gel" refers to gel material having amorphous structural composition. In
an embodiment, the number of multifunctional silica based nanoparticle in
a gram of multifunctional silica based nanoparticle gel can be difficult
to accurately determine. However, the following provides some guidance.

[0059] Let's assume amorphous silica gel (completely dehydrated) including
about 10 nm size (diameter) inter-connected particles as our test
material. One could roughly estimate number of particles per gram of
material in the following way:

Mass (m) of a single particle=density of the particle (d)×volume
of the particle (v)

D=2.648 gm/cm3 (approx) V=(4/3)Pi (π)(r)3 Where r is the
radius of the particle. π=3.14; r=(10/2 nm)=5 nm=5×10-7 cm.
If we plug in these numbers, v=5.23×10-19 cm3 Then
m=(5.23×10-19 cm3)(2.648 gm/cm3) Or,
m=1.38×10-18 gm

[0060] The multifunctional nanoparticle/nanogel product contains two
active components, DMDS and first component (e.g., Cu) and a second
component (e.g., DMDS). Experimentally, we can load about 33 to 45 wt %
of Cu in silica nanoparticle material (measured by ICP-AAS analysis; ICP
stands for Inductively Coupled Plasma-Atomic Absorption Spectroscopy).
For example, Cu loading is about 33% in Cu loaded silica nanogel material
synthesized in acidic ethanol-water mixture containing ethanol (95%) up
to 45.5% of total volume. Cu loading is about 45% in Cu loaded silica
nanoparticle material synthesized only in acidic water. Roughly one Cu
can hold at least one DMDS molecule. These estimates can be applied to
the first component and the second component.

[0061] An embodiment of the multifunctional silica based nanoparticle and
gel are described in PCT Patent Application US 2009/006496 entitled
"Silica-based Antibacterial and Antifungal Nanoformulation", which is
incorporated herein by reference. In addition, methods of making an
embodiment of the multifunctional silica based nanoparticle and gel are
described in the aforementioned PCT Patent Application.

[0062] In general, the precursor material to make the multifunctional
silica based nanoparticles and gel can be made by mixing a silane
compound (e.g., alkyl silane, tetraethoxysilane, tetramethoxysilane,
sodium silicate, or a silane precursor that can produce silicic acid or
silicic acid like intermediates and a combination of these silane
compounds) with a first component precursor compound in an acid medium
(e.g., acidic water) that may contain an alcohol such as ethanol. After
mixing for a period of time (e.g., about 30 minutes to a few hours), a
mixture including silica nanoparticles loaded with the first component
(also referred to as a "loaded silica nanoparticle") is formed. After the
loaded silica nanoparticle are formed, the medium can be brought to a pH
of about 7 and held for a time period (e.g., a few hours to a day) to
form a precursor material that includes a loaded silica nanoparticle gel,
where the nanoparticles are inter-connected. This process can be
performed using a single reaction vessel or can use multiple reaction
vessels.

[0063] Once the loaded silica nanoparticle is made, the multifunctional
silica based nanoparticles and gel can be formed. The loaded silica
nanoparticle can be disposed in a reaction vessel in an aqueous reaction
medium (e.g., acidic water) or can be dried and mixed as a powder. The
second component (e.g., DMDS) is also added to the reaction vessel that
includes the aqueous reaction mixture or the dry precursor material. The
ratio of the amount of precursor material and the second component (dry)
can be about 1 to 1. This mixture is mixed for a period of time (e.g.,
from minutes to hours) to form the multifunctional silica based
nanoparticles and gel. The multifunctional silica based nanoparticles and
gel can be separated (e.g., centrifuge) from the aqueous solution and
dried (e.g., air dried). The mixture does not require any additional
purification, although further purification and processing can be
performed. This process can be performed using a single reaction vessel
or can use multiple reaction vessels and can be performed at ambient
temperature and pressure.

[0064] In a particular embodiment, the second component is DMDS and can be
added under mechanical stirring after Cu loaded silica nanoformulation is
prepared. However, DMDS can be added anytime during the nanoformulation
preparation process. In an embodiment, about 100 micrograms of DMDS is
added to about 45 g equivalent of Cu.

[0065] In another embodiment of the present disclosure the multifunctional
silica based nanoparticles gel can be formed using a second silane
compound, where the addition of the second silane compound improves the
uniformity of the plant surface coverage. During the step when the silane
compound is added, the second silane compound can also be added. The
second silane compound can include compounds such as alkyl silanes. The
second silane compound can be about 0.01 to 30% or about 10 to 30% weight
of the silane compound. The resulting silane mixture can include the
first component and/or the second component, such as those described
above. The nanoparticle is the same or similar to the nanoparticle
described above and herein. It should be noted that an objective in this
embodiment is to tailor nanoparticle/nanogel surface hydrophilicity or
hydrophobicity to further improve adherence property of nanoformulations.
For example citrus leaves are waxy (hydrophobic). To improve adherence of
nanoformulation to waxy surface via hydrophobic-hydrophobic interaction,
silica nanoparticle/nanogel material can be further modified with a
hydrophobic silane reagent such methyl- or propyl- or butyl silane.

[0066] As mentioned above, embodiments of the present disclosure are
effective for the treatment of diseases affecting plants such as citrus
plants and trees. In addition, embodiments of the present disclosure can
be effective as a protective barrier against phloem-feeding ACPs as it
uniformly covers the plant surface (e.g., leaf surface). In particular,
embodiments of the present disclosure can be used to combat citrus canker
and greening diseases (HLB). The design of the multifunctional silica
based nanoparticle or gel facilitate uniform plant surface coverage or
substantially uniform plant surface coverage. In an embodiment, the
multifunctional silica based nanoparticle or gel that is applied to
plants can have a superior adherence property in various types of
exposure to atmospheric conditions such as rain, wind, snow, and
sunlight, such that it is not substantially removed over the time frame
of the release of the first and/or second components. In an embodiment,
the multifunctional silica based nanoparticle or gel has a reduced
phytotoxic effect on plants and reduced environmental stress due to
minimal Cu content.

[0067] Embodiments of the present disclosure can applied on the time
frames consistent with the release of the first and second components,
and these time frames can include from the first day of application to
about a week, about a month, about two months, about three months, about
four months, about five months, about six months, about seven month, or
about eight months.

[0068] A specific embodiment of the multifunctional silica based
nanoparticle or gel can include a multifunctional dimethyl disulfide
(DMDS) and copper co-loaded silica based nanoparticle or nanogel
(DMDS-CuSiNP/NG), which can be used to combat citrus canker and greening
diseases is disclosed. When the composition is applied to citrus plants,
it is effective for simultaneously controlling citrus canker and citrus
greening diseases in a single application during one citrus growing
season.

EXAMPLES

[0069] Now having described the embodiments of the present disclosure, in
general, the examples describe some additional embodiments of the present
disclosure. While embodiments of the present disclosure are described in
connection with examples and the corresponding text and figures, there is
no intent to limit embodiments of the present disclosure to these
descriptions. On the contrary, the intent is to cover all alternatives,
modifications, and equivalents included within the spirit and scope of
embodiments of the present disclosure.

Example 1

CuSiNG Material and Properties

[0070] Laboratory-based experimental data confirmed the following: (i)
much improved antibacterial properties of CuSiNG material in comparison
to controls, Kocide® 3000 (a DuPont product; Cu hydroxide material)
and Cu sulfate, (ii) exceptionally strong adherence property to the
citrus leaf surface in comparison to controls and (iii) improved surface
coverage, uniform throughout, upon spray application. Based on materials
characterization data (High Resolution Transmission electron Microscopy
and Selected Area Electron Diffraction patterns), it is confirmed that Cu
is present in silica nanogel (SiNG) matrix in two different forms and in
two different oxidation states, crystalline Cu oxide (Cu+1 state) and
amorphous Cu complex (Cu+2 state). The CuSiNG is thus a unique,
nanotechnology-enabled engineered nanomaterial. The disc diffusion assay
confirmed that the CuSiNG material has the ability to diffuse out from
the application location. This diffusion property will have strong impact
in protecting rapidly expanding young fruit and leaf surfaces.

Example 2

DMDS Loaded CuSiNG in Solution

[0071] In the present disclosure, DMDS and Cu co-loaded silica nanogel
(DMDS-CuSiNG) based materials and related formulations are prepared.
Multidisciplinary research is used to develop and study DMDS loaded
materials, conduct a number of laboratory based bioassays to test the
efficacy and perform field study to evaluate the efficacy in controlling
HLB and citrus canker diseases.

[0072] The loading of DMDS in CuSiNG has been studied in solution state
(as synthesized CuSiNG liquid formulation) and characterized by the Gas
Chromatography-Mass Spectrometry (GC-MS). In solution state, loading of
DMDS into CuSiNG material was carried out by directly adding DMDS into
the aqueous reaction mixture that contains CuSiNG material. Stirring was
continued to ensure uniform mixing of DMDS with the CuSiNG material. The
reaction medium composition greatly facilitated direct loading of DMDS
into CuSiNG material. After 24 hrs, DMDS-CuSiNG material was centrifuged
and air-dried for more than seven days. We were able to smell strong
sulphur odor.

[0073]FIG. 1A is GC-MS spectra of DMDS used as a control under conditions
wherein the silica nanogel (SiNG) does not contain copper. FIG. 1B is a
GC-MS spectra of DMDS-CuSiNG material. A DMDS odor from the DMDS-CuSiNG
powder is discernable even after seven days. For GC-MS sample
preparation, spectroscopy grade chloroform was added to the powder and
DMDS. GC-MS data of DMDS (control) and chloroform extract of DMDS-CuSiNG
are shown in FIG. 1A and FIG. 1B, respectively. Characteristic molecular
peak for DMDS at 93 (m/z) along with other peaks for its fragmented
structure is shown in FIG. 2 where the GC-MS spectra of DMDS-SiNG
material products were found in both cases, confirming the presence of
DMDS in DMDS-CuSiNG sample.

[0074] Similar experiment was also carried out with SiNG (instead of
CuSiNG). After 3 days, we were not able to detect characteristic DMDS
odor from DMDS-SiNG material and GC-MS study showed no noticeable DMDS
characteristic peaks as shown in FIG. 2. These results suggest that
Cu2+ ions play a critical role with DMDS loading and retention.

Example 3

Loading of DMDS into CuSiNG in Dry State

[0075] Loading of DMDS in dry state using lyophilized CuSiNG powder was
carried out by adding DMDS (100 μl neat) directly to CuSiNG (150 mg of
vacuum dried powder) sample in a 20 mL glass vial. For a quick comparison
purposes, we took 100 μl neat DMDS in another 20 mL glass vial
(control). Both vials were kept side-by-side inside a laboratory fume
hood to allow DMDS to evaporate at the same rate. After 3 days, we were
able to smell strong DMDS odor from the DMDS-treated CuSiNG sample only.

[0076] Subsequently, chloroform is added to both vials and results are
shown in FIGS. 3a and 3b. GC-MS spectra of DMDS added to dry CuSiNG
lyophilized powder is shown in FIG. 3A. In FIG. 3B a control extract of
DMDS is taken and performed GC-MS. As expected, characteristic DMDS peaks
were obtained from DMDS-CuSiNG sample (FIG. 3A) and no such peaks from
the control (FIG. 3B). The above preliminary experiments thus confirmed
that CuSiNG material is able to load, retain and slowly release DMDS. A
synthesis method for preparation of a silica matrix with embedded
metallic particles is reported in U.S. Pat. No. 6,548,264 to Tan et al.,
U.S. Pat. No. 6,924,116 to Tan et al., and U.S. Pat. No. 7,332,351 to Tan
et al., which are incorporated herein by reference. The synthesis of
CuSiNG is disclosed in International Patent Application No.
PCT/US2009/006496 filed Dec. 10, 2009 and is incorporated herein by
reference.

Example 4

Characterization of DMDS-CuSiNG Material

[0077] The following material characterization techniques were used to
characterize the DMDS-CuSiNG material of the present disclosure. First a
GC-MS study will qualitatively confirm loading of DMDS in CuSiNG and SiNG
materials. Second Quartz Crystal Microbalance (QCM) based sensing study
will confirm loading and release of DMDS in real-time. Considering
practical application of DMDS-CuSiNG material in the field, it is
desirable to perform quantitative study to monitor DMDS loading/release
processes in real-time. Therefore QCM based sensing technology will be
adapted for quantitative measurements of DMDS loading/release and
determine kinetics. The sensitivity of QCM technique is reported at the
parts per billion level by J. W. Gardner et al, "A brief-history of
electronic noses." Sensors and Actuators B-Chemical 1994, 18, (1-3),
211-220. The characterization of DMDS-CuSiNG clarifies the nature of
interaction of DMDS with the CuSiNG material. Our goal is to investigate
the physico-chemical environment around DMDS and the role of Cu in DMDS
adsorption.

[0078] Briefly, the experimental setup includes using a sample of CuSiNG
material that is spray-coated onto QCM sensor followed by exposure to
DMDS in a closed chamber. It is expected that with time the resonating
frequency of the QCM will continue to decrease as more and more DMDS is
loaded into the CuSiNG material. Once equilibrium is reached, no further
frequency drop will take place. The sensor is removed from the chamber
and monitoring of the DMDS release process with time is observed. We
expect to observe increase in frequency as more and more DMDS is
released. Similar experiments will be performed for the SiNG material, as
a control.

[0080] Unlike SiNG, the physico-chemical environment of DMDS in CuSiNG is
expected to be somewhat different due to presence of Cu (II) ions.
Preliminary results suggest that characteristic odor DMDS does not change
over time which indicates that DMDS is non-reactive to Cu(II) ions.
However, the DMDS electron-rich sulfur atom has the ability to weakly
interact with electron-deficient Cu(II) ion. This could further
facilitate adsorption of DMDS into CuSiNG. Thorough FT-IR studies are
performed to understand the nature of intermolecular interactions that
exist between DMDS and SiNG. In addition, thermogravimetric analysis
(TGA), calorimetry and QCM sensing studies to obtain DMDS loading/release
characteristics (isotherms) against CuSiNG and SiNG materials will be
used to determine the role of Cu II ions in DMDS loading. A comparative
isotherm data analysis along with FT-IR analysis will reveal the effect
of Cu in DMDS loading/release process.

Example 6

DMDS Loading into CuSiNG Through Nanoscale Manipulation

[0081] The manipulation of the molecular environment around DMDS at the
nanoscale level was conducted to improve DMDS loading efficiency into
CuSiNG and SiNG materials.

[0082] First hybrid silica nanogel (HSiNG) and Cu loaded HSiNG (CuHSiNG)
materials were synthesized. A combination of two inexpensive silica
precursors were used during the HSiNG synthesis consisting of a silane
based ester and an alkyl based (e.g. methyl or propyl) silane. The
rationale of introducing small alkyl chain polymers into the silica
matrix is that it will improve interaction with DMDS via intermolecular
hydrophobic-hydrophobic interaction.

[0083] A series of experiments were performed by varying the ratio of
these two silica precursors to optimize loading of both Cu and DMDS into
HSiNG material. Both the DMDS-HSiNG and DMDS-HCuSiNG materials are
systematically characterized. DMDS loading efficiency to HSiNG and
HCuSiNG materials will be evaluated and results will be compared with
DMDS-CuSiNG material. For bioassays and field trials, only one
DMDS-CuSiNG material that has maximum loading of Cu and DMDS will be
selected, the two active components responsible for preventing canker and
HLB diseases, respectively.

[0084] Characterization: Cu loading efficiency will be quantitatively
determined by the atomic absorption spectroscopy (AAS) whereas DMDS
loading/release kinetics will be determined by the QCM study. In
addition, several material characterization techniques such as TEM/HRTEM
(size and morphology), SAED (crystallinity), XPS (identification of Cu
oxidation states), XRD (bulk crystallinity), SEM-EDAX (elemental analysis
for estimating Cu to Si ratio), BET (surface area/porosity measurements)
and FTIR (studying interaction of DMDS as well as Cu with silica matrix)
will be used for systematic characterization of HSiNG and CuHSiNG
materials. Further improvement of DMDS loading into HSiNG and HCuSiNG is
expected due to additional hydrophobic-hydrophobic intermolecular
interactions between the methyl groups of DMDS and alkyl groups of silane
precursor.

Example 7

Comparing Efficacy of DMDS-CuSiNG and DMDS-HCuSiNG

[0085] Laboratory bioassays for efficacy evaluation of DMDS loaded
materials include, disc diffusion assays against X. alfalfae and
Olfactometer bioassays against ACPs and the disc diffusion assay. To test
anti-bacterial activity of the DMDS-CuSiNG and DMDS-HCuSiNG materials, a
known `disc diffusion assay` method is used.

[0086] Briefly, appropriate dilution of overnight grown X. alfalfae
culture (200 μl of about 106 cfu/ml based on dilution of the 0.5 Mc
Farland standard) will be spread on nutrient agar plates (90 mm) to
achieve a confluent lawn of growth. Comparative analysis of growth
inhibition by different concentrations of the test materials (DMDS-CuSiNG
and DMDS-HcuSiNG) using DMDS-SiNG and DMDS-HSING as negative controls,
and Cu sulfate and Kocide® 3000 as positive controls will be
performed to find out the difference in minimum inhibitory concentration
ranges under the test conditions. The same volume containing different
concentrations of each formulation will be applied per disc to avoid any
variation of unequal diffusion perimeter on the agar plate. The level of
anti-bacterial ability will be determined by measuring the zone of
inhibition after 24 hrs of incubation at 30° C. The minimum
inhibitory concentration (MIC) of all the above mentioned compounds,
including DMDS-CuSiNG and DMDS-HCuSiNG, will also be determined by
spreading appropriate dilution of X. alfalfae culture on nutrient agar
plates containing log2 (two fold) serial dilutions of the test
compound(s) to correlate the results of the zone of inhibition in the
disc diffusion test and to refine the concentration of DMDS-CuSiNG and
DMDS-HCuSiNG necessary for effective killing of the pathogens.

[0087] All the test plates will be incubated for longer periods beyond 24
hours to detect any delayed growth of the test organism by the formation
of isolated colonies in the zone of inhibition or on the dilution series
agar plates at the MIC level. This will tentatively indicate the
difference in stability between the DMDS-CuSiNG (or DMDS-HCuSiNG) and
Kocide® 3000 or Cu sulfate under the test conditions, which will be
an indirect evaluation of the difference in Cu release kinetics of the
DMDS-CuSiNG (or DMDS-HCuSiNG) and the Kocide® 3000. We will also
generate dose-response plots wherever appropriate. Briefly, dosage of the
Cu compound will be varied and the relative response or percentages of
disease control to those dosages will be determined.

[0088] Statistical analysis: We will test the significance of
anti-bacterial activity by different statistical analyses such as ANNOVA
discussed in Design and Analysis of Experiments by Hinkelmann, K.;
Kempthorne, O., Wiley: 2008; Vol. I and II (Second ed.). Diameter of
inhibition zone with all the respective anti-bacterial materials at
different concentrations and incubation times will be measured to use in
the statistical analysis, assessing the suitability of CuSiNG and
H--CuSiNG as a better anti-bacterial material with respect to the
controls.

[0089] The benefits of applying nanotechnology in citrus research are
enormously high. Silica nanogel matrix provides a unique environment for
co-hosting Cu and DMDS, making DMDS-CuSiNG nanomaterial multifunctional
for combating both citrus canker and greening diseases. Recent studies
suggest that silica nanogel is able to slowly spread out from the point
of application in moist environment. The impact would be tremendously
high as this CuSiNG will form a uniform film-like coating on the surface
over time. We identify two major advantages of silica nanogel film; (i)
ability to protect rapidly growing young fruit and leaf surfaces and (ii)
ability to serve as protective barrier for phloem-feeding ACPs.

[0091] Due to nanoscale engineering, the CuSiNG of the present disclosure
has the following advantages over the existing Cu based compounds:
uniform coverage of plant surface because of ultra-small particle size,
better adherence property due to gel-like nanostructure, sustained
(long-term) Cu release profile, better control on Cu release rate
(adjustable "soluble" to "insoluble" Cu ratio), more
antibacterial/antifungal activity with less amount of Cu content, reduced
phytotoxic effect because of adjustable "soluble" to "insoluble" Cu ratio
and environment-safe due to less Cu content, no harmful by-product
formation, water-based synthesis, utilization of excess CuSiNG as plant
nutrient and minimal possibility of having elevated local Cu
concentration that could cause environmental toxicity.

[0092] The synthesis protocol has the following advantages: (i)
simplicity, (ii) water-based, (ii) scalable to field applications, (iii)
single-pot synthesis method, requiring no purification steps and (v)
concentrated CuSiNG material could be easily diluted for field
application. A non-technical person can do this task by adding an
appropriate amount of water, thus reducing shipping costs. The method
also uses inexpensive raw chemicals and is easily produced in a
cost-effective manner.

[0093] It should be noted that ratios, concentrations, amounts, and other
numerical data may be expressed herein in a range format. It is to be
understood that such a range format is used for convenience and brevity,
and thus, should be interpreted in a flexible manner to include not only
the numerical values explicitly recited as the limits of the range, but
also to include all the individual numerical values or sub-ranges
encompassed within that range as if each numerical value and sub-range is
explicitly recited. To illustrate, a concentration range of "about 0.1%
to about 5%" should be interpreted to include not only the explicitly
recited concentration of about 0.1 wt % to about 5 wt %, but also include
individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges
(e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. In
an embodiment, the term "about" can include traditional rounding
according to significant figures of the numerical value. In addition, the
phrase "about `x` to `y`" includes "about `x` to about `y`".

[0094] It should be emphasized that the above-described embodiments of the
present disclosure are merely possible examples of implementations, and
are set forth only for a clear understanding of the principles of the
disclosure. Many variations and modifications may be made to the
above-described embodiments of the disclosure without departing
substantially from the spirit and principles of the disclosure. All such
modifications and variations are intended to be included herein within
the scope of this disclosure.

Patent applications by Swadeshmukul Santra, Orlando, FL US

Patent applications by University of Central Florida Research Foundation, Inc.